U.S. patent number 7,589,075 [Application Number 10/466,263] was granted by the patent office on 2009-09-15 for use of an adenosine a3 receptor agonist for inhibition of viral replication.
This patent grant is currently assigned to Can-Fite Biopharma Ltd.. Invention is credited to Pnina Fishman, Kamel Khalili.
United States Patent |
7,589,075 |
Fishman , et al. |
September 15, 2009 |
Use of an adenosine A3 receptor agonist for inhibition of viral
replication
Abstract
The present invention concerns the use of an active ingredient
selected from the group consisting of agonists of the adenosine
receptor system, for inhibiting viral replication in cells. In
particular, the invention provides a composition and method for
inhibiting viral replication in cells, the method comprising
presenting to the cells an effective amount of the active
ingredient. According to one embodiment, the adenosine agonist is
an A3 receptor agonist (A3RAg). The invention is particularly
useful, for although not limited to, inhibiting the replication of
HIV virus in human cells.
Inventors: |
Fishman; Pnina (Herzliya,
IL), Khalili; Kamel (Merion, PA) |
Assignee: |
Can-Fite Biopharma Ltd. (Petach
Tikva, IL)
|
Family
ID: |
22994269 |
Appl.
No.: |
10/466,263 |
Filed: |
January 13, 2002 |
PCT
Filed: |
January 13, 2002 |
PCT No.: |
PCT/IL02/00028 |
371(c)(1),(2),(4) Date: |
January 15, 2004 |
PCT
Pub. No.: |
WO02/055085 |
PCT
Pub. Date: |
July 18, 2002 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20040106572 A1 |
Jun 3, 2004 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60261659 |
Jan 16, 2001 |
|
|
|
|
Current U.S.
Class: |
514/45;
514/263.36; 514/263.3; 514/263.23; 514/263.35 |
Current CPC
Class: |
A61P
31/18 (20180101); A61K 31/52 (20130101); A61K
31/7076 (20130101); A61K 31/00 (20130101); A61K
31/522 (20130101); A61P 31/12 (20180101) |
Current International
Class: |
A61K
31/52 (20060101); A61K 31/522 (20060101); A61K
31/7076 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 94/21195 |
|
Sep 1994 |
|
WO |
|
WO 95/02604 |
|
Jan 1995 |
|
WO |
|
WO 99/06053 |
|
Feb 1999 |
|
WO |
|
WO 99/20284 |
|
Apr 1999 |
|
WO |
|
WO 01/19360 |
|
Mar 2001 |
|
WO |
|
Other References
Zhou QY, Li C, Olah ME, Johnson RA, Stiles GL, Civelli O.
"Molecular cloning and characterization of an adenosine receptor:
the A3 adenosine receptor." Proc Natl Acad Sci U S A. Aug 15,
1992;89(16):7432-6. cited by examiner .
Thomas CA, Weinberger OK, Ziegler BL, Greenberg S, Schieren I,
Silverstein SC, El Khoury "Human immunodeficiency virus-1 env
impairs Fc reception-mediated phagocytosis via a cyclic adenosine
monophosphate-dependent mechanism." J.Blood. Nov 1,
1997;90(9):3760-5. cited by examiner .
Osborn et. al. Tumor Necrosis Factor .alpha. and Interleukin 1
Stimulate the Human Immunodeficiency Virus Enhancer by Activation
of the Nuclear Factor .kappa.B. Proceedings of the National Academy
of Sciences of the United States of America, vol. 86, No. 7. (Apr.
1, 1989), pp. 2336-2340. cited by examiner .
Sajjadi, et al. Inhibition of TNF-a Expression by Adenosine: Role
of A3 Adenosine ReceptorsJ Immunol, 1996, 156: 3435-3442. cited by
examiner .
Shneyvays, et al. Induction of Apoptosis in Cardiac Myocytes by an
A3 Adenosine Receptor Agonist. Exp Cell Res. 1998; 243:383-397.
cited by examiner .
9-Nitocamptothecin Inhibits HIV-1 Replication in Human Peripheral
Bloood Lymphocytes: A Potential Alternative for HIV-Infection/AIDS
Therapy. J Med. Virol. 2001; 64:238-244. cited by examiner .
Hasko, et al. Adenosine Receptor Agonists Difierentially Regulate
II-10, TNF-a, and Nitric Oxide Production in RAW 264.7 Macrophages
and in Endotoxemic Mice. The Iournal of Immunology, 1996,157:
4634-4640. cited by examiner .
Bouma, Maarten; "Differential Regulatory Effects of Adenosine on
Cytokine Release by Activated Human Monocytes"; Journal of
Immunology. 1994; 4159-4167. cited by other .
Gartner, Suzanne; "Virus Isolation and Production", Basic Virologic
Techniques. Immunology Department, Primate Research Institute, New
Mexico State University. 1990, 53-70. cited by other .
Gilbertsen, R.; "Adenosine and Adenosine Receptors in Immune
Function. Minireview and meeting report", Agents and Actions. 1987,
vol. 22, 1/2, 91-98. cited by other .
Gonzalez, Fernando; "Activation of Early Events of Mitogenic
Response by a P.sub.2y Purinoceptor with Covalently Bound
3'-O-(4-benzoyl)-benzoyladenosine 5'-triphosphate", Proc. Natl.
Acad. Sci. Dec. 1990 vol. 87, 9717-9721. cited by other .
Linden, Joel; "Structure and Function of A.sub.1 Adenosine
Receptors"; The FASEB Journal. Sep. 1991, vol. 5, 2668-2675. cited
by other .
Pastan, Ira; "Role of Cyclic Nucleotides in Growth Control",
Laboratory of Molecular Biology. Nation Cancer Institute, National
Institutes of Health. 1975, 491-522. cited by other .
Rozengurt, Enrique; "Adenosine Receptor Activation in Quiescent
Swiss 3T3 Cells"; Experimental Cell Research. 1982, vol. 139;
71-78. cited by other .
Sandber, G.; "Regulation of Thymocyte Proliferation: Effects of
L-Alanine, Adenosine and Cyclic AMP in vitro". Thymus. 1981; vol.
3,63-75. cited by other .
Soderback, U.; "Anti-aggregatory Effects of Physiological
Concentrations of Adenosine in Human Whole Blood as Assessed by
Filtragometry", Clinical Science. 1991, vol. 81, 691-694. cited by
other .
Stiles, Gary; "Adenosine Receptors and Beyond: Molecular Mechanisms
of Physiological Regulation", Clinical Research. 1989, 12-18. cited
by other .
Stolfi, Robert; "Modulation of 5-Fluorouracil-induced Toxicity in
Mice with Interferon of with the Interferon Inducer,
Polyinosinic-Polycytidylic Acid", Cancer Research. Feb. 1983,
561-566. cited by other .
Csaba Szabo et al., "Suppression of macrophage inflammatory protein
(MIP)-1.alpha. production and collagen-induced arthritis by
adenosine receptor agonists", British Journal of Pharmacology,
(1998) 125, 379-387. cited by other .
Calabrese et al., "Safety of antitumour necrosis factor (anti-TNF)
therapy in patients with chronic viral infections: Hepatitis C,
hepatitis B, and HIV infection" Ann Rheum Dis. 63:18-24 (2004).
cited by other .
Sha et al., "Effect of etanercept (Enbrel) on interleukin 6, tumor
necrosis factor alpha, and markers of immune activation in HIV
infected subjects receiving interleukin 2" AIDS Res Hum
Retroviruses. 18(9):661-5 (2002). cited by other .
Brabers et al., "Role of the proinflammatory cytokines TNF-.alpha.
and IL-1.beta. in HIV-associated dementia Nottet" European Journal
of Clinical Investigation. 36:447-458 (2006). cited by other .
Hermida-Escobedo et al., "A double-blinded clinical trial to assess
the effect of pentoxifylline in the inhibition of tumor necrosis
factor production in patients with AIDS" Int Conf AIDS. Jul. 7-12;
11: 123 (1996). cited by other .
Okeoma et al., "APOBEC3 inhibits mice mammary tumor virus
replication in vivo," Nature. 445:927-930 (2007). cited by other
.
Siekmann and Lawson, "Notch signaling limits angiogenic cell
behavior in developing zebra fish arteries," Nature. 445:781-784
(2007). cited by other .
Piccirillo et al., "Bone morphogenetic proteins inhibit the
tumorigenic potential of human brain tumor-initiating cells,"
Nature. 444:761-765 (2006). cited by other .
Lane et al., "TNF-.alpha. inhibits HIV-1 replication in peripheral
blood monocytes and alveolar macrophages by inducing the production
of RANTES and decreasing C-C chemokine receptor 5(CCR5) expression"
J. Immunol. 163:3653-3661 (1999). cited by other .
Li et al. "The relationship between tumor necrosis factor and human
immunodeficiency virus gene expression in lymphoid tissue" J.
Virol. 71:7080-7082 (1997). cited by other .
Herbein et al. "Tumor Necrosis Factor Alpha inhibits entry of human
immunodeficiency virus-type 1 into primary human macrophages: a
selective role for the 75-kilodalton receptor" J. Virol.
70:7388-7397 (1996). cited by other .
Mestan et al., "Antiviral effects of recombinant tumor necrosis
factor in vitro" Nature 323:816-819 (1986). cited by other .
Ito et al., "Antiviral effects of recombinant human tumor necrosis
factor" Lympokine Res. 6:309-318 (1987). cited by other .
Baharav et al., "Antiinflammatory Effect of A3 Adenosine Receptor
Agonists in Murine Autoimmune Arthritis Models," J Rheumatol
32469-476 (2005). cited by other .
Fishman et al., "The A3 Adenosine Receptor as a New Target for
Cancer Therapy and Chemoprotection," Exp Cell Res 269:230-236
(2001). cited by other .
Fishman et al., "Evidence for involvement of Wnt signaling pathway
in IB-MECA mediated suppression of melanoma cells," Oncogene
21:4060-4064 (2002). cited by other .
van Troostenburg et al., "Tolerability, pharmacokinetics and
concentration-dependent hemodynamic effects of oral CF101, and A3
adenosine receptor agonist, in healthy young men" Int. J. Clin.
Pharmacol. Ther. 42:534-542 (2004). cited by other.
|
Primary Examiner: Mosher; Mary E
Assistant Examiner: Snyder; Stuart W
Attorney, Agent or Firm: Browdy and Neimark, P.L.L.C.
Claims
The invention claimed is:
1. A method for inhibiting replication of a virus in cells
comprising contacting the cells with an effective amount of at
least one adenosine A3 receptor agonist (A3RAg).
2. The method of claim 1, wherein the virus is an HIV.
3. The method of claim 1, wherein said A3RAg is a nucleotide
derivative of the following general formula (I): ##STR00006##
wherein R.sub.1 is alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl
or a group of the following general formula (II): ##STR00007## in
which: Y is oxygen, sulfur, or CH.sub.2; X.sub.1 is H, alkyl,
R.sup.aR.sup.bNC(.dbd.O)--or HOR.sup.c--, wherein R.sup.a and
R.sup.b may be the same or different and are selected from the
group consisting of hydrogen, amino, a substituted or unsubstituted
alkyl, haloalkyl, aminoalkyl, protected-aminoalkyl, and cycloalkyl,
or are joined together to form a heterocyclic ring containing two
to five carbon atoms, and R.sup.c is selected from the group
consisting of alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl,
and cycloalkyl; X.sub.2 is H, hydroxyl, alkylamino, alkylamido or
hydroxyalkyl; X.sub.3 and X.sub.4 each independently are hydrogen,
hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy,
nitrilo, nitro, trifluoro, aryl, alkaryl, thio, ester, thioester,
ether, thioether, --OCOPh, or --OC(.dbd.S)OPh or both X.sub.3 and
X.sub.4 are oxygen connected to >C.dbd.S to form a 5-membered
ring, or X.sub.2 and X.sub.3 form the ring of formula (III):
##STR00008## where R' and R''' are independently alkyl; R.sub.2 is
selected from the group consisting of hydrogen, halo, alkylether,
amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio,
alkenyl; alkynyl, thio, and alkylthio; and R.sub.3 is a
--NR.sub.4R.sub.5 group with R.sub.4 being hydrogen, alkyl,
substituted alkyl or aryl-NH--C(Z)--, with Z being O, S, or
NR.sup.a with R.sup.a having the above meanings, and R.sub.5 is a
group selected from the group consisting of heteroaryl-NR.sup.a--
C(Z)--, heteroaryl-C(Z)--, alkaryl-NR.sup.a--C(Z)--,
alkaryl-C(Z)--, aryl-NR--C(Z)--and aryl-C(Z)--, with R.sup.a and Z
having the above defined meanings; or, when R.sub.4 is a hydrogen,
R.sub.5 is R-- or S-1-phenylethyl, benzyl, phenylethyl or anilide,
all of the above optionally substituted in one or more positions
with a substituent selected from the group consisting of alkyl,
amino, halo, haloalkyl, nitro, hydroxyl, acetamido, alkoxy, and
sulfonic acid or a salt thereof; or when R.sub.4 is
benzodioxanemethyl, fururyl, L-propylalanylaminobenzyl,
.beta.-alanylaminobenzyl, T-protected-.beta.-alanylaminobenzyl,
phenylamino, carbamoyl, phenoxy or cycloalkyl, then R.sub.5 is a
group of the following formula: ##STR00009## or a suitable salt of
the compound defined above.
4. The method of claim 3, wherein said A3RAg is a nucleoside
derivative of the general formula (IV): ##STR00010## wherein
X.sub.1, R.sub.2 R.sub.4 and R.sub.5 are as defined in claim 3.
5. The method of claim 4, wherein the active ingredient is an
N.sup.6-benzyladenosine-5'-uronamide.
6. The method of claim 5, wherein said A3RAg is selected from the
group consisting of N.sup.6-2-(4-aminophenyl)ethyladenosine
(APNEA), N.sup.6-(4-amino-3-iodobenzyl)
adenosine-5'-(N-methyluronamide) (AB-MECA) and 1-deoxy
-1-{6-[({3-iodophenyl}methyl)
amino]-9H-purine-9-yl}-N-methyl-.beta.-D-ribofuranuronamide
(IB-MECA) and
2-chloro-N.sup.6-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide
(Cl-IB-MECA).
7. The method of claim 6, wherein said A3RAg is IB-MECA or
Cl-IB-MECA.
8. A method for inhibiting replication of a virus in cells of an
individual in need of treatment for viral infection, comprising
administering to the individual an effective amount of at least one
adenosine A3 receptor agonist (A3RAg).
9. The method of claim 8, wherein the virus is an HIV.
10. The method of claim 8, wherein the at least one A3RAg is a
nucleotide derivative of the following general formula (I):
##STR00011## wherein R.sub.1 is alkyl, hydroxyalkyl, carboxyalkyl
or cyanoalkyl or a group of the following general formula (II):
##STR00012## in which: Y is oxygen, sulfur, or CH.sub.2; X.sub.1 is
H, alkyl, R.sup.aR.sup.bNC(.dbd.O)-- or HOR.sup.c--, wherein
R.sup.a and R.sup.b may be the same or different and are selected
from the group consisting of hydrogen, amino, a substituted or
unsubstituted alkyl, haloalkyl, aminoalkyl, protected-aminoalkyl,
and cycloalkyl, or are joined together to form a heterocyclic ring
containing two to five carbon atoms, and R.sup.c is selected from
the group consisting of alkyl, amino, haloalkyl, aminoalkyl,
BOC-aminoalkyl, and cycloalkyl; X.sub.2 is H, hydroxyl, alkylamino,
alkylamido or hydroxyalkyl; X.sub.3 and X.sub.4 each independently
are hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy,
carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, ester,
thioester, ether, thioether, --OCOPh, or --OC(.dbd.S)OPh or both
X.sub.3 and X.sub.4 are oxygen connected to >C.dbd.S to form a
5-membered ring, or X.sub.2 and X.sub.3 form the ring of formula
(III): ##STR00013## where R' and R'' are independently alkyl;
R.sub.2 is selected from the group consisting of hydrogen, halo,
alkylether, amino, hydrazido, alkylamino, alkoxy, thioalkoxy,
pyridylthio, alkenyl; alkynyl, thio, and alkylthio; and R.sub.3 is
a --NR.sub.4R.sub.5 group with R.sub.4 being hydrogen, alkyl,
substituted alkyl or aryl-NH--C(Z)--, with Z being O, S, or
NR.sup.a with R.sup.a having the above meanings, and R.sub.5 is a
group selected from the group consisting of heteroaryl-NR.sup.a--
C(Z)--, heteroaryl-C(Z)--, alkaryl-NR.sup.a--C(Z)--,
alkaryl-C(Z)--, aryl-NR--C(Z)-- and aryl-C(Z)--, with R.sup.a and Z
having the above defined meanings; or, when R.sub.4 is a hydrogen,
R.sub.5 is R-- or 5-1-phenylethyl, benzyl, phenylethyl or anilide,
all of the above optionally substituted in one or more positions
with a substituent selected from the group consisting of alkyl,
amino, halo, haloalkyl, nitro, hydroxyl, acetamido, alkoxy, and
sulfonic acid or a salt thereof; or when R.sub.4 is
benzodioxanemethyl, fururyl, L-propylalanylaminobenzyl,
.beta.-alanylaminobenzyl, T-protected-.beta.-alanylaminobenzyl,
phenylamino, carbamoyl, phenoxy or cycloalkyl, then R.sub.5 is a
group of the following formula: ##STR00014## or a suitable salt of
the compound defined above.
11. The method of claim 10, wherein the at least one A3RAg is a
nucleoside derivative of the general formula (IV): ##STR00015##
wherein X.sub.1, R.sub.2 R.sub.4 and R.sub.5 are as defined in
claim 10.
12. The method of claim 11, wherein the at least one A3RAg is an
N.sup.6-benzyladenosine-5'-uronamide.
13. The method of claim 12, wherein the at least one A3RAg is
selected from the group consisting of
N.sup.6-2-(4-aminophenyl)ethyladenosine (APNEA),
N.sup.6-(4-amino-3-iodobenzyl) adenosine-5'-(N-methyluronamide)
(AB-MECA) and
1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-.beta-
.-D-ribofuranuronamide (IB-MECA) and
2-chloro-N.sup.6-(3-iodobenzyl)-adenosine-5'-N-methyl-uronamide
(Cl-IB-MECA).
14. The method of 3, wherein said suitable salt of the compound is
a triethylammonium salt.
15. The method of claim 13, wherein the at least one A3RAg is
IB-MECA or Cl-IB-MECA.
16. The method of claim 8, wherein said suitable salt of the
compound is a triethylammonium salt.
Description
FIELD OF THE INVENTION
The present invention is generally in the field of anti-infectives
and more specifically it concerns pharmaceutical compositions and
medical uses for inhibiting viral replication inside cells.
PRIOR ART
The following is a list of prior art, which is considered to be
pertinent for describing the state of the art in the field of the
invention. Acknowledgement of these references herein will be made
by indicating the number from their list below within brackets.
1. Linden J. The FASEB J. 5:2668-2676 (1991);
2. Stiles G. L. Clin. Res. 38:10-18 (1990);
3. Stolfi R. L., et al. Cancer Res. 43:561-566 (1983);
4. Soderback U. et al. Clin. Sci. 81:691-694 (1994);
5. Gilbertsen R. B. Agents actions 22:91-98 (1987);
6. Bouma M. G. et al. J. Immunol. 153: 4159-4168 (1994);
7. Rozengurt E. Exp. Cell Res. 139:71-78 (1982);
8. Gonzales F. A., et al., PNAS USA 87:9717-9721 (1990);
9. Sandberg G. and Fredholm B. B., Thymus 3:63-75 (1981);
10. Pastan I. H. et al. Annu. Rev. Biochem. 44:491-495 (1975);
11. R. Cole and J. de Vellis. 1997. In: Protocols for neural cell
culture. S. Fedoroff and A. Richardson (Eds.) Human Press, Totowa,
N.J., pp. 117-130.
12. S. Gartner and M. Popovic. 1990. In: Techniques on HIV
research. A Aldovini and B. D. Walker (Eds.) Stockton Press. New
York, N.Y., pp. 53-70.
13. V. W. Yong and J. P. Antel, 1997. In: Protocols for neural cell
culture. S. Fedoroff and A. Richardson (Eds.) Humana Press, Totowa,
N.J., pp. 157-172.
14. U.S. Pat. No. 5,688,774.
15. U.S. Pat. No. 5,773,423.
16. U.S. Pat. No. 6,048,865.
17. WO 95/02604.
18. WO 99/20284.
19. WO 99/06053
BACKGROUND OF THE INVENTION
Human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) are
retroviruses that cause acquired immunodeficiency syndrome (AIDS)
in humans. AIDS results from low levels of CD4-positive
T-lymphocytes in HIV-infected individuals.
HIV-1 infects T-lymphocytes, monocytes/macrophages, dendritic cells
and microglia. All of these cells express the surface glycoprotein
CD4 which serves as a receptor for HIV-1 and HIV-2. Efficient entry
of HIV-1 into target cells is dependent upon binding of the viral
envelope glycoprotein gp120 to CD4. In addition, several chemokine
receptors function as HIV co-receptors and determine efficient
infection of various cell types with HIV-1 strains. After binding,
the HIV-1 envelope glycoproteins mediate fusion of viral and host
cell membranes to complete the entry process. Once inside the
cells, a process of viral replication occurs and through a budding
process replicated viruses are released from infected cells, This
eventually leads to cytolytic destruction of the infected cells.
This sequence is repeated many times thereby significantly reducing
the number of the target cells in the body, which is a severe and
life-threatening material state often giving rise to eventual death
of the infected individual.
Adenosine is a purine nucleoside present in plasma and other
extracellular fluids. It is released into the extracellular space
by various cell types and exerts an effect or other cells by
binding to G-protein associated receptors on the cell
membrane.sup.(1-2). The interaction of adenosine with its receptors
initiates signal transduction pathways, progressing mainly the
adenylate cyclase effector system, which utilizes cAMP as a second
messenger. G-protein associated adenosine receptors are classified
into four groups referred to as A1, A2a, A2b and A3. A1 and A3
receptors are coupled with G.sub.i proteins and thus inhibit
adenylate cyclase leading to a decrease in the level of
intracellular cAMP. The A2a and A2b receptors are coupled to
G.sub.s proteins and thus activates adenylate cyclase, thereby
increasing cAMP levels.sup.(3).
Among the physiological effects of extracellular adenosine are
inhibition of cytokine release, inhibition of platelet aggregation,
induction of erythropoietin production and modulation of lymphocyte
function.sup.(4-6). Adenosine is also involved in the modulation of
some central nervous system (CNS) functions, in wound healing, in
diuresis and in controlling pain. Adenosine is capable of inducing
proliferation in a wide range of normal cell types.sup.(7-10).
SUMMARY OF THE INVENTION
The present invention is based upon the finding that adenosine
receptor agonists inhibit viral replication inside cells. Thus, in
accordance with the invention, there is provided a method for
inhibiting viral replication in cells, comprising presenting to the
cells an effective amount of at least one adenosine A3 receptor
agonist (A3RAg).
The agonist according to the invention is either a full or partial
agonist of the adenosine A.sub.3 receptor. As used herein, a
compound is a "full agonist" of an adenosine A.sub.3 receptor if it
is able to fully inhibit adenylate cyclase (A.sub.3), a compound is
a "partial agoinist" of an adenosine A.sub.3 receptor if it is able
to partially inhibit adenylate cyclase (A.sub.3).
Also provided by the invention are pharmaceutical compositions for
inhibiting viral replication inside cells, comprising an effective
amount of said at least one A3RAg, as well as the use of said
active ingredient (i.e. the A3RAg) for the manufacture of such a
pharmaceutical composition.
The invention is particularly useful, although not limited to,
inhibiting the replication of HIV virus in human cells.
DETAILED DESCRIPTION OF THE INVENTION
The pharmaceutically or therapeutically "effective amount" for
purposes herein is determined by such considerations as may be
known in the art. The amount must be effective to achieve the
desired therapeutic effect, which depends on the type and mode of
treatment. As is clear to the artisan, the effective amount should
be effective to reduce the rate of viral replication inside cells,
to reduce the level of viral particles in clinical samples, or to
obtain an improvement in the condition of an individual having a
viral infection, to obtain an improvement or elimination of
symptoms or any other indicators acceptable as appropriate measures
by those skilled in the art. An example of an effective amount is a
daily administration of an A3RAg within the range of between about
1 .mu.g/kg body weight and about 10 mg/kg body weight. Such an
amount of A3RAg is typically administered in a single daily dose
although at times a daily dose may be divided into several doses
administered throughout the day or at times several daily doses may
be combined into a single dose to be given to the patient once
every several days, particularly if administered in a sustained
release formulation.
By one embodiment, the active ingredient is a nucleoside
derivative. By the term "nucleoside" it is meant any compound
comprising a sugar, preferably ribose or deoxyribose, or a purine
or pyrimidine base or a combination of a sugar with a purine or
pyrimidine base preferably bound to one another through a
N-glycosyl link. The term "nucleoside derivative" will be used to
denote herein, a naturally occurring nucleoside, a synthetic
nucleoside or a nucleoside, which underwent chemical modifications
by way of one or more insertions, deletions, exocyclic or
endocyclic substitutions of one or more groups thererin or
conformational modifications which provide a derivative with the
desired biological effect.
According to one embodiment of the invention, the active ingredient
is a nucleoside derivative of the following general formula
(I):
##STR00001## wherein R.sub.1 is alkyl, hydroxyalkyl carboxyalkyl or
cyanoalkyl or a group of the following general formula (II):
##STR00002## in which: Y is oxygen, sulfur of carbon atoms; X.sub.1
is H, alkyl R.sup.aR.sup.bNC(.dbd.O)-- or HOR.sup.c--, wherein
R.sup.a and R.sup.b may be the same or different and are selected
from the group consisting of hydrogen, amino; or a substituted or
unsubstituted alkyl, haloalkyl, aminoalkyl, BOC-aminoalkyl, and
cycloalkyl or are joined together to form a heterocyclic ring
containing two to five carbon atoms, and R.sup.c is selected from
the group consisting of alkyl, amino, haloalkyl, aminoalkyl,
protected aminoalkyl (e.g.-BOC aminoalkyl), and cycloalkyl; X.sub.2
is H, hydroxyl, alkylamino, alkylamido or hydroxyalkyl; X.sub.3 and
X.sub.4 each independently are hydrogen, hydroxyl, amino, amido,
azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro,
aryl, alkaryl, thio, ester, thioester, ether, thioether, --OCOPh,
--OC(.dbd.S)OPh or both X.sub.3 and X.sub.4 are oxygen connected to
>C.dbd.S to form a 5-membered ring, or X.sub.2 and X.sub.3 form
the ring of formula (III):
##STR00003## where R' and R'' are independently a lower alkyl;
R.sub.2 is selected from the group consisting of hydrogen, halo,
alkylether, amino, hydrazido, alkylamino, alkoxy, thioalkoxy,
pyridylthio, alkenyl; alkynyl, thio, and alkylthio; and R.sub.3 is
an --NR.sub.4R.sub.5 group, wherein R.sub.4 is a hydrogen or a
group selected from alkyl, substituted alkyl or aryl-NH--C(Z)-,
with Z being O, S, or NR.sup.a with R.sup.a having the above
meanings, and R.sub.5, is a group selected from
heteroaryl-NR.sup.a--C(Z)-, heteroaryl-C(Z)-,
alkaryl-NR.sup.a--C(Z)-, alkaryl-C(Z)-, aryl-NR--C(Z)- and
aryl-C(Z)-, R.sup.a and Z having the above defined meanings; or,
when R.sub.4 is a hydrogen, R.sub.5 is R-- or S-1-phenylethyl,
benzyl, phenylethyl or anilide, all of the above optionally
substituted in one or more positions with a substituent selected
from the group consisting of alkyl, amino, halo, haloalkyl, nitro,
hydroxyl, acetoamido, alkoxy, and sulfonic acid or a salt thereof;
or when R.sub.4 is benzodioxanemethyl, fururyl,
L-propylalanylaminobenzyl, .beta.-alanylamino-benzyl,
T-BOC-.beta.-alanylamino-benzyl, phenylamino, carbamoyl, phenoxy or
cycloalkyl,
R.sub.5 is a group of the following formula:
##STR00004##
or a suitable salt of the compound defined above, e.g. a
triethylammonium salt thereof.
The active ingredient is preferably a nucleoside derivative of the
general formula (IV):
##STR00005## wherein X.sub.1, R.sub.2 R.sub.4 and R.sub.5 are as
defined above and
Preferred active ingredients according to this embodiment of the
invention may generally be referred to as
N.sup.6-benzyladenosine-5'-uronamides and derivatives thereof found
to be A3-selective adenosine receptor agonists. Examples for such
derivatives are N.sup.6-2-(4-aminophenyl)ethyladenosine (APNEA),
N.sup.6-(4-amino-3-iodobenzyl) adenosine-5'-(N-methyluronamide)
(AB-MECA) and
1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-.-
beta.-D-ribofuranuron-amide, the latter also referred to in the art
as N.sup.6-3-idobenzyl-5'-methylcarboxamidoadenosine or
N.sup.6-(3-idobenzyl)adenosine-5'-N-methyl-uronamide and herein
above and below by the abbreviation IB-MECA. A chlorinated
derivative of IB-MECA (R.sub.2.dbd.Cl) also forms part of this
group and is referred to herein as Cl-IB-MECA, IB-MECA and
Cl-IB-MECA being currently preferred.
According to another embodiment of the invention, the active
ingredient may be adenosine derivative generally referred to as
N.sup.6-benzyl-adenosine-5'-alkyluronamide-N.sup.1-oxide or
N.sup.6-benzyladenosine-5'-N-dialyluron-amide-N.sup.1-oxide.
Some of the above defined compounds and their synthesis procedure
may be found in publications 14 to 19 listed above, incorporated
herein by reference.
The hydrocarbon chains used herein may include straight or branched
chains. In particular, the term "alkyl" refers to monovalent
straight, branched of cyclc alkyl groups preferably having from
1-20 carbon atoms, more preferably 1-10 carbon atomes ("lower
alkyl") and most preferably 1 to 6 atoms. This term is exemplified
by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, n-hexyl, and the like.
The terms "alkylene" and "lower alkylene" refer to divalent
radicals of the corresponding alkane. Further, as used herein,
other moieties having names derived from alkanes, such as alkoxyl,
alkanoyl, alkenyl, cycloalkenyl, etc. when modified by "lower",
have carbon chains of ten or less carbon atoms. In those cases
where the minimum number of carbons are greater than one, e.g.,
alkenyl (minimum of two carbons) and cycloalkyl, (minimum of three
carbons), it is to be understood that "lower" means at least the
minimum number of carbons.
As used herein, the term "substituted alkyl" refers to an alkyl
group, having from 1 to 4 substituents, and preferably 1 to 3
substituents as defined above. As used herein, other moieties
having the prefix "substituted" are intended to include one or more
of the substituents listed above.
As used herein, the term "alkoxy" refers to the group "alkyl-O--" ,
where alkyl is as defined above.
As used herein, the term "alkenyl" refers to alkenyl groups
preferably having from 2 to 10 carbon atoms and more preferably 2
to 6 carbon atoms and having at least 1 and preferably from 1-2
sites of alkenyl unsaturation while the term "alkynyl" refers to
alkynyl groups preferably having from 2 to 10 carbon atoms and more
preferably 2 to 6 carbon atoms and having at least 1 and preferably
from 1-2 sites of alkynyl unsaturation.
As used herein, the term "aryl" refers to an unsaturated aromatic
carbocyclic group of from 6 to 14 carbon atoms having a single ring
(e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl
or anthryl). Preferred aryls include phenyl, naphthyl and the like.
Unless otherwise stated by the definition for the aryl substituent,
such aryl groups can optionally be substituted with from 1 to 5
substituents and preferably 1 to 3 substituents such as those
provided above.
As used herein, the term "cycloalkyl" refers to cyclic alkyl groups
of from 3 to 12 carbon atoms having a single cyclic ring or
multiple condensed rings. Such cycloalkyl groups include, by way of
example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclooctyl, and the like, or multiple ring structures
such as adamantanyl, and the like.
As used herein, the term "heteroaryl" refers to an aromatic
carbocyclic group of from 1 to 15 carbon atoms and 1 to 4
heteroatoms selected from the group consisting of oxygen, nitrogen
and sulfur within at least one ring (if there is more than one
ring). Unless otherwise stated such heteroaryl groups can be
optionally substituted with from 1 to 5 substituents and preferably
1 to 3 substituents as indicated above.
As to any of the above groups that contain 1 or more substituents,
it is understood, of course, that such groups do not contain any
substitution or substitution patterns which are sterically
impractical and/or synthetically non-feasible.
At times, the above defined A3RAg, being the active ingredient, may
contain a protecting groups or blocking groups. The term
"protecting group" or "blocking group" refers to any group which
when bound to one or more hydroxyl, amino or carboxyl groups of the
compounds prevents reaction from occurring at these groups and
which protecting group can be removed by conventional chemical or
enzymatic steps to reestablish the hydroxyl, amino or carboxyl
group. Preferred removable amino blocking groups include
conventional substituents such as t-butyoxycarbonyl (t-BOC)
(indicated above) as well as others such as, benzyloxycarbonyl
(CBZ), and the like which can be removed by conventional conditions
compatible with the nature of the product.
The A3RAg in accordance with the invention may be as defined above
or may be in the form of salts or solvates thereof, in particular
physiologically acceptable salts and solvates thereof. Further,
when containing one or more asymmetric carbon atoms, the active
ingredient may include isomers and diastereoisomers of the above
active ingredients or mixtures thereof.
Pharmaceutically acceptable salts of the above active ingredients
include those derived from pharmaceutically acceptable inorganic
and organic acids. Examples of suitable acids include hydrochloric,
hydrobromic, sulphoric, nitric, perchloric, fumaric, maleic,
phosphoric, glycollic, lactic, salicylic, succinic,
toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
formic, benzoic, malonic, naphthalene-2-sulfonic and
benzenesulfonic acids.
The A3RAg may be administered as a non-active substance (e.g.
pro-drug) and be made active only upon further modification/s by a
natural process at a specific site in the subject. In any case, the
derivative will be such that the therapeutic functionality of the
pharmaceutical composition of the invention, is preserved. Such
pro-drugs are also encompassed by the term "active ingredient" as
used herein. Similarly, the term "A3RAg" should be understood as
encompassing pro-drugs which, although a priori, lack the agonistic
activity, become active in vivo.
To choose an adenosine A3 receptor agonist to be used in accordance
with the invention, candidate components may be screened for such
compounds which have an ability to inhibit viral replication in a
manner resembling that of IB-MECA or CI-IB-MECA. A suitable screen
is an in vitro assay of the kind described in the Experiments
Results Section below. However, a variety of other assays known per
se may also be used.
The pharmaceutical composition of the invention may comprise the
A3RAg as such, but may be combined with other ingredients which may
be a pharmaceutically acceptable carrier, diluent, excipient,
additive and/or adjuvent, as known to the artisan, e.g. for the
purposes of adding flavors, colors, lubrication or the like to the
pharmaceutical composition. Evidently, the pharmaceutically
acceptable carrier/s, diluent/s, excipient/s, additive/s employed
according to the invention generally refer to inert, non-toxic
solid or liquid fillers, diluents or encapsulating materials which
preferably do not react with the compounds within the composition
of the invention.
Many A3RAgs are bioavailable when orally administered. Thus,
depending on the active ingredient, the pharmaceutical composition
of the invention may be formulated for oral administration. Such an
oral composition may further comprise a pharmaceutically acceptable
carrier, diluent, excipient, additive or adjuvant suitable for oral
administration.
The pharmaceutical compositions of the invention are administered
and dosed in accordance with good medical practice, taking into
account the clinical condition of the individual patient, the site
and method of administration, scheduling of administration,
patient's age, sex, body weight and other factors known to medical
practitioners.
The composition of the invention may be administered in various
ways. It can be administered orally, subcutaneously or parenterally
including intravenous, intraarterial, intramuscular,
intraperitoneally or by intranasal administration, as well as by
intrathecal and infusion techniques known to the man versed in the
art.
The treatment has an overall length contingent to the length of the
disease process and active agent effectiveness. The therapeutic
regimen may involve single doses or multiple doses over a period of
several days or more.
When administering the compositions of the present invention
parenterally, it will generally be formulated in a unit dosage
injectable form (solution, suspension, emulsion). The
pharmaceutical formulation suitable for injection includes sterile
aqueous solutions or dispersions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
The carrier employed can be a solvent or dispersing medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, lipid polyethylene glycol and the
like), suitable mixtures thereof and vegetable oils.
Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil,
soybean oil, corn oil, sunflower oil, or peanut oil and ester, such
as isopropyl myristate, may also at times be used as solvent
systems for the active ingredient.
Additionally, various additives which enhance the stability,
sterility and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating agents and
buffers can be added. Prevention of the action of microorganisms
can be ensured by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid and the
like.
For the purpose of oral administration, the active ingredient may
be formulated in the form of tablets, suspensions, solutions,
emulsions, capsules, powders, syrups and the like, are usable and
may be obtained by techniques well known to the pharmacists.
The present invention will now be described by way of example with
reference to the experimental results below and to the accompanying
Figures. It is to be understood, that the terminology which has
been used is intended to be in the nature of words of description
rather than limitation.
While the foregoing description describes in detail only a few
specific embodiments of the invention, it will be understood by
those skilled in the art that the invention is not limited thereto
and that other variations in form and details may be possible
without departing from the scope and spirit of the invention herein
disclosed.
EXPERIMENTAL RESULTS
Materials and Methods:
Preparation of Primary Human Fetal Astrocytes and Microglia.
Purified primary human fetal astrocytes and microgial cells were
prepared from 16 to 20 week old human fetal brain tissue by a
modified procedure based on the methods of Cole and de
Vellis.sup.11, and Yong and Ante.sup.13. Brain tissue was washed in
ice-cold Hank's Balanced Salt Solution (HBSS) containing the
antibiotics gentamycin and amphotericin B. Blood vessels and
meninges were removed and the tissue was minced into small pieces.
After mincing, the tissue was enzymatically dissociated by
incubation in 0.05% trypsin and mechanically disrupted by passing
several times over a 75 .mu.m nylon mesh filter. The resulting
single cell suspension was washed, pelleted and plated at a density
of 2-10.times.10.sup.6 cells per 162 cm.sup.2 flask in DMEM:F12
containing 10% fetal calf serum, insulin, gentamycin, and
L-glutamine. After 7-10 days of growth, microglial cells were
isolated by placement on rotary shaker at 200 rpm in a 37.degree.
C. incubator overnight. The non-adherent cells were removed and
allowed to attach to a new flask for 1 to 3 h. Following
attachment, the cells were washed and refed with media containing
10% fetal calf serum, insulin, gentamycin, L-glutamine, and NI
supplement. Astrocytes were subcultured from adherent cells in
media containing 15% fetal calf serum, insulin, gentamycin, and
L-glutamine and contaminating microglia were removed by repeated
rotary shaking. Cultured astrocytic and microglial cells were
plated at a density of 2.5.times.105 per well into 6 well plates
for subsequent infection.
Preparation of HIV-1 Virus
Brain derived primary HIV-1 isolates SF162 and JR-FL were cultured
in human peripheral blood mononuclear cells (PBMC) essentially as
described by Gartner and Popovic.sup.12. PBMC were isolated from
human buffy coat by fico gradient and plated at a density of
2.5.times.10.sup.6 per ml in RPMI containing 10% fet calf serum and
gentamycin. Cells were stimulated by the addition of 5 ug/ml
phytohemagglutinin (PHA) for 48 h. After stimulation, cells were
infected will either SF162 or JR-FL and cultured for 7 to 10 days
until high titres of HIV-1 we detected in the supernatant by p24
ELISA assay. When viral production was optimal, the cells were
pelleted, the supernatant containing HIV-1 was aliquoted and stored
at -70.degree. C. until use. P24 ELISA assay was performed on an
aliquoted stock to determine the viral titre.
Infection of Primary Human Fetal Astrocytic and Microglial Cells
and Treatment with CI-IB-MECA.
2.5.times.10.sup.5 microglial or astrocytic cells were plated per
well into 6-we plates. The next day, cells were washed and refed
with fresh medium. 2.times.10.sup.4 p2units of either SF 162 or
JR-FL virus was added per well in a total of 1 ml of vir inoculum.
In control experiments, the virus was not added. Cells were
incubated with virus overnight at 37.degree. C., washed extensively
with PBS, and refed with 2 .pi. fresh medium. Cultures were treated
with IB-MECA or CI-IB-MECA at concentration of 0.01 .mu.M every 24
hours. 500 .mu.l of medium were removed at the indicated times
following infection and stored at -70.degree. C. for later
analysis. Each time medium was removed, a volume amount of fresh
medium was added. In control experiments IB-MECA and Cl-IB-MECA
were omitted.
p24 ELISA Assay.
ELISA assay to detect the HIV-1 viral core protein, p24, was
performed of 50 .mu.l of the collected supernatant utilizing the
commercially available p24 ELISA Kit (NEN/Dupont) according to the
manufacturer's instructions.
Results
A seen in Tables 1 to 3, the amount of p24 protein present in
culture medium collected from HIV infected cells is significantly
reduced in HIV infected cells treated with IB-MECA (HIV and
IB-MECA) or Cl-IB-MECA (HIV and CI IB-MECA) in comparison to
controls not related with either IB-MECA or Cl-IB-MECA, (HIV).
Table 1 shows the effect of IB-MECA and Cl-IB-MECA on HIV
replication in JR-FL infected astroglial cells, wherein p 24
protein (pg/mL) was measured in medium from cell cultures 5 days
after HIV infection.
Table 2 shows the effect of IB-MECA and Cl-IB-MECA on HIV
replication in SF162 infected astroglia, wherein p 24 protein
(pg/mL) was measured as indicated above.
Table shows the effect of IB-MECA and Cl-IB-MECA on HIV replication
in SF126 infected microglia/SF, wherein p 24 protein (pg/mL) was
measured in medium from cell cultures 5 days and 10 days after HIV
infection.
TABLE-US-00001 TABLE 1 Astroglia/JR-FL Treatment p24 pg/mL Day 5 No
HIV 12.64 HIV 22.83 HIV and IB-MECA 0.01 3.02 HIV and Cl-IB-MECA
0.01 8.45
TABLE-US-00002 TABLE 2 Astroglia/SF-162 Treatment p24 pg/mL Day 5
No HIV -12.96 HIV 313.38 IB-MECA 0.01 137.58 Cl-IB-MECA 0.01
288.77
TABLE-US-00003 TABLE 3 Microglia/SF p24 pg/mL Day 5 after infection
Day 10 after infection No HIV -12.64 -12.64 HIV 267.99 209.18
IB-MECA 0.01 81.33 62.79 IB-MECA 0.1 82.29 54.80 Cl-IB-MECA 0.01
127.03 111.05 Cl-IB-MECA 0.0.1 10.81 80.37
* * * * *